Compositions and method for treating infection in cattle and swine

ABSTRACT

Novel formulations combining a non-steroidal anti-inflammatory drug (NSAID) such as flunixin, with a fluorinated chloramphenicol or thiamphenicol derivative antibiotic such as florfenicol are disclosed. Methods for using such formulations in the treatment and prevention of infectious diseases of bovines and swine, including bovine respiratory disease and swine respiratory disease, are also disclosed.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent claims priority as a continuation of U.S. patent applicationSer. No. 10/441,392 (filed May 20, 2003; now abandoned), which, in turn,claims benefit of priority to U.S. Provisional Patent Application60/382,015 (filed May 20, 2002, now expired). The entire text of each ofthe above-referenced patent applications is incorporated by referenceinto this patent.

FIELD OF THE INVENTION

The invention relates to compositions and methods for the treatment ofinfections in animals. More particularly, the invention relates to acomposition containing both an antibiotic and a non-steroidalanti-inflammatory drug for use in the treatment of infections in animalssuch as cattle and swine.

BACKGROUND OF THE INVENTION

All references cited herein are hereby incorporated in their entirety byreference.

Bovine respiratory disease (BRD) occurs in both dairy and beef cattleand is one of the leading causes of economic loss to the cattle industrythroughout the world. These economic losses are due to excessivemortality, reduced weight gains as well as treatment and preventioncosts. BRD is often referred to as the “bovine respiratory diseasescomplex” due to the multifactorial etiology.

The cost of death losses due to respiratory diseases vary around theworld. Death losses in the U.S. are estimated to approach $1 billionannually. Losses in various European countries range from $75 to $120million. Cattle with clinical or sub-clinical BRD do not gain weight orproduce milk as well as healthy animals. Beef cattle with BRD gain lessweight, have reduced feed efficiency and often produce a lower gradecarcass at slaughter. Perino L. J., Apley M., Bovine RespiratoryDisease, in CURRENT VETERINARY THERAPY 4 (FOOD ANIMAL PRACTICE), 4^(TH)ED. 446-455 (Howard J. L., Smith R. A., eds., 1999). A directcorrelation between pulmonary lesions observed at slaughter and reducedweight gains has been established in cattle with sub-clinicalinfections. Whittem T. E. et al., J. Am. Vet. Med. Assoc., 209:814-818(1996).

In addition to the production losses associated with mortality andmorbidity, significant costs are associated with the treatment of BRDdue to the costs of various therapeutic agents and the labor required toadminister these agents, along with the extra labor to isolate andobserve these animals.

The pathogenesis of BRD is thought to be due to the interaction ofenvironmental and physiological stresses coupled with infectious agents.Mannheimia (Pasteurella) haemolytica, Pasteurella multocida andHaemophilus somnus are considered part of the normal flora of the bovineupper respiratory tract. When environmental and physiological stressfactors reduce the natural resistance and inhibit the pulmonary defensemechanisms these organisms proliferate and colonize the lowerrespiratory tract. In addition, various bovine viruses such asinfectious bovine rhinotracheitis virus (IBRV), bovine viral diarrheavirus (BVDV), bovine respiratory syncytial virus (BRSV) andparainfluenza 3 virus (PI-3) are known to have immunosuppressive effectsin the lung.

Similarly, swine respiratory disease (SRD) also has a multifactionaletiology. Bacterial infections caused by P. multocida, H. parasuis,Bordetella bronchiseptica, Actinobacillus pleuropneumoniae,Streptococcus suis, Salmonella cholerasuis and Mycoplasma sp. can resultin respiratory disease in swine, resulting in significant economiclosses. Stresses such as crowding, mixing and moving of pigs andtransient viral infections can contribute to the intensification of thedisease.

These organisms can stimulate an excessive inflammatory process in thelungs by producing various toxins that stimulate the release of variouscytokines, which up-regulate the inflammatory process. M. haemolytica,considered the most virulent of these organisms, also produces aleukotoxin that inhibits phagocytosis by leukocytes thus furtherenhancing its ability to colonize the lower respiratory tract. Thisprocess often results in a bacterial bronchopneumonia.

The pulmonary damage that results in death or morbidity is due to theexcessive inflammatory response to the invading pathogens. Damage tohost tissues occur as neutrophils, pulmonary alveolar macrophages andnatural killer cells destroy infected cells. As cell membranes aredamaged, arachidonic acid is released. Arachidonic acid is the substratefor the formation of various prostaglandins and other eicosanoids. Therelease of these biological active substances is critical to driving theinflammatory response that results in pulmonary lesions. Mosier D. A.,Vet. Clin. North Am. Food Animal Prac., 13:483-493 (1997).

In general, therapy for BRD should be directed at achieving thefollowing goals:

1. Controlling the infection—In animals where the infectious process ishalted early, the need for repeat treatment is significantly reduced(see Apley M. D. & Fajt V. R., Vet. Clin. North Am. Food Anim. Prac.,14:291-313 (1998). The selection of the appropriate antimicrobialcompound should be based on the antimicrobial sensitivity of theorganism involved, the levels of the antimicrobial agent in therespiratory tract, ease of administration, the potential for injectionsite tissue damage and a dosing regime that minimizes the pain andstress associated with treatment.

2. Minimize the pulmonary damage—As the level of inflammation andsubsequent pulmonary damage increases, the probability of repeat therapyincreases and the rate of weight gain decreases. Lekeux P., BovinePractitioner, 29:71-75 (1995); Scott P. R., J. Dairy Sci., 76(2):414-420(1993).

3. Reduce pyrexia—Controlling the infection and reducing theinflammation will reduce the pyrexia (fever) thus increasing thepotential for recovery. The feeling of well-being that accompanies thereduction of pyrexia may also improve the intake of nutrients bysuppressing inappetence associated with disease and pyrexia.

For years antimicrobial therapy has been the mainstay of BRD therapy.There are many effective microbial agents currently available for thetreatment of BRD. NUFLOR, an injectable formulation of the broadspectrum antibiotic florfenicol, has emerged as one of the leadingantibiotics on a global basis. It is indicated for the treatment andcontrol of BRD associated with M. haemolytica, P. multocida and H.somnus as well as for the prevention of respiratory disease in cattle athigh risk of developing BRD associated with these bacteria. NUFLOR isalso indicated for the treatment of bovine interdigital phlegmon(footrot, acute interdigital necrobacillosis, infectious pododermatitis)associated with Fusobacterium necrophorum and Bacteroidesmelaninogenicus. NUFLOR may be administered subcutaneously as well asintramuscularly.

The pathogenesis of BRD involves the development of a significantinflammatory process in the lungs and the subsequent development ofpulmonary lesions, often leading to pulmonary consolidation. The degreeof this inflammatory process can determine whether the disease resultsin mortality, a chronic “poor doer” or the animal recovers uneventfully.Various anti-inflammatory agents have been investigated regarding theirability to reduce the pyrexia, lung consolidation and weight lossassociated with BRD.

The use of corticosteroids is generally contraindicated as ancillarytherapy for BRD due to their ability to cause serious immunosuppression.The use of non-steroidal anti-inflammatory drugs (NSAIDs) in conjunctionwith antibiotics, however, has been shown to be of benefit in thetreatment of bovine respiratory disease. The nonsteroidal,anti-inflammatory agent flunixin meglumine has been demonstrated to beeffective in rapidly reducing pyrexia associated with BRD. Flunixin hasalso been demonstrated to reduce pulmonary consolidation and the needfor re-treatment with antibiotics.

Flunixin meglumine is the active ingredient in FINADYNE and BANAMINE(both available from Schering-Plough Animal Health Corporation, Union,N.J.). It has emerged as one of the leading NSAIDs for adjunctivetherapy of BRD.

Flunixin meglumine has been studied extensively in regard to its use inconjunction with antibiotics for the treatment of BRD. While it iswidely used for this indication, it has not been used in combination inthe same formulation with florfenicol because the primary route ofadministration of flunixin is intravenous and florfenicol isadministered intramuscularly or subcutaneously. Moreover, florfenicolformulations have been designed to provide prolonged blood levels of theantibiotic and flunixin would not be expected to have adequatebioavailability in such formulations. In addition, there have beenconcerns as to whether florfenicol and flunixin would be compatible insuch formulations.

Flunixin meglumine has been used in conjunction with oxytetracycline,and products containing both flunixin meglumine and oxytetracycline arecommercially available in Europe. However, such combination productsrequire once per day administration for 3 to 5 days. Furthermore,resistance to the antibiotic oxytetracycline has become commonplace inregard to bacterial pathogens, including those commonly associated withBRD.

Accordingly, there is a need for conveniently administered, stablecompositions that can control and prevent the infection and minimize theinflammation associated with bovine respiratory disease and otherinfectious diseases, while minimizing the pain and stress to the animalassociated with treatment and the potential for injection site tissuedamage.

SUMMARY OF THE INVENTION

The present invention fulfills this need by providing improvedcompositions and methods for the treatment of bovine respiratory diseaseand other infections of cattle and swine.

The present invention relates to a composition for the treatment ofmicrobial infection in an animal comprising flunixin or one of itspharmaceutically acceptable salts and a compound of Formula I:

wherein R is a member selected from the group consisting of methyl orethyl or a halogenated derivative thereof, dihalogenodeuteriomethyl,1-halogeno-1-deuterioethyl, 1,2-dihalogeno-1-deuterioethyl, azidomethyland methylsulfonylmethyl;

each of X and X′ is a member independently selected from the groupconsisting of NO₂, SO₂R₁, SOR₁, SR₁, SONH₂, SO₂NH₂, SONHR₁, SO₂NHR₁,COR₁, OR₁, R₁, CN, halogen, hydrogen, phenyl, and phenyl substituted byhalogen, NO₂, R₁, PO₂R₁, CONHR₁, NHR₁, NR₁R₂, CONR₁R₂, OCOR₁, or OR₁,wherein each of R₁ and R₂ is a member independently selected from thegroup consisting of methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl,isobutyl and phenyl;

and Z is hydrogen or an acyl group of a hydrocarboncarboxylic acidhaving up to 16 carbon atoms or an acyl group of anaminohydrocarboncarboxylic acid having up to 12 carbon atoms; and thepharmaceutically acceptable salts of said acyl groups.

In a preferred embodiment, the composition for the treatment ofmicrobial infection in an animal comprises (a) florfenicol; (b) flunixinor one of its pharmaceutically acceptable salts; and (c) from about 5%to about 80% of an aprotic polar solvent.

The present invention also relates to a method of treating a microbialinfection in an animal comprising the step of subcutaneouslyadministering to an animal in need of such treatment a therapeuticallyeffective amount of a composition comprising flunixin or one of itspharmaceutically acceptable salts and a compound of Formula I.

In a preferred embodiment, the microbial infection is selected from thegroup consisting of bovine respiratory disease, swine respiratorydisease, footrot, acute mastitis, pinkeye, metritis and enteritis.

The present invention also relates to a method of preventing a microbialinfection in an animal susceptible to such an infection comprising thestep of subcutaneously administering to an animal susceptible to such aninfection a prophylactic amount of a composition comprising flunixin orone of its pharmaceutically acceptable salts and a compound of FormulaI.

In a preferred embodiment, the microbial infection is bovine respiratorydisease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the mean plasma concentration (+/−SD) offlorfenicol vs. time after a single subcutaneous injection of NUFLOR(florfenicol) or a composition of the present invention containing bothflorfenicol and flunixin meglumine.

FIG. 2 is a graph showing the mean plasma concentration of flunixin vs.time after parenteral administration of BANAMINE/FINADYNE flunixinmeglumine or a florfenicol-flunixin meglumine combination composition ofthe present invention.

FIG. 3 is a graph showing the effect of flunixin within aflorfenicol-flunixin meglumine combination composition of the presentinvention.

FIG. 4 is a graph showing the effect of treatment with aflorfenicol-flunixin meglumine combination composition of the presentinvention on antipyretic response in cattle with BRD.

FIG. 5 is a graph showing the effect of treatment with aflorfenicol-flunixin meglumine combination composition of the presentinvention on lung consolidation score.

FIG. 6 is a graph showing the effect of treatment with aflorfenicol-flunixin meglumine combination composition of the presentinvention on the clinical success rate.

DETAILED DESCRIPTION OF THE INVENTION

When flunixin is administered subcutaneously in a combinationformulation with an antibiotic, its bioavailability is significantlyreduced. One would expect that this would result in less clinicalefficacy when administered in this manner compared to simultaneousadministration by the intravenous route.

It has surprisingly been found, however, that, despite the reducedbioavailability of subcutaneously administered flunixin meglumine, thesame dose of flunixin meglumine when administered in certainformulations containing a fluorinated chloramphenicol or thiamphenicolanalog/derivative antibiotic, is as clinically efficacious as the sameamount of flunixin meglumine administered intravenously in conjunctionwith a separate subcutaneous injection of the antibiotic.

As used herein, the following terms, unless otherwise indicated, shallbe understood to have the following meanings:

“Acyl” means an H—C(O)—, alkyl-C(O)—, alkenyl-C(O)—, alkynyl-C(O)—,cycloalkyl-C(O)—, cycloalkenyl-C(O)—, or cycloalkynyl-C(O)— group inwhich the various groups are as previously described. The bond to theparent moiety is through the carbonyl. Preferred acyls contain a loweralkyl. Non-limiting examples of suitable acyl groups include formyl,acetyl, propanoyl, 2-methylpropanoyl, butanoyl and cyclohexanoyl.

“Alkyl” means an aliphatic hydrocarbon group, which may be straight orbranched, comprising from 1 to about 20 carbon atoms in the chain.Preferred alkyl groups contain from 1 to about 12 carbon atoms in thechain. More preferred alkyl groups contain from 1 to about 6 carbonatoms in the chain. Branched means that one or more lower alkyl groups,such as methyl, ethyl or propyl, are attached to a linear alkyl chain.“Lower alkyl” means a group having from 1 to about 6 carbon atoms in thechain, which may be straight or branched. The term “substituted alkyl”means that the alkyl group may be substituted by one or moresubstituents which may be the same or different.

“Aryl” means an aromatic monocyclic or multicyclic ring systemcomprising about 6 to about 14 carbon atoms, preferably about 6 to about10 carbon atoms. The aryl group can be optionally substituted with oneor more “ring system substituents,” which may be the same or different,and are as defined herein.

“Alkoxy” means an alkyl-O— group in which the alkyl group is aspreviously described. Non-limiting examples of suitable alkoxy groupsinclude methoxy, ethoxy, isopropoxy, and n-butoxy. The bond to theparent moiety is through the ether oxygen.

“Azido” refers to an —N₃ group.

“Halo” and “halogeno” mean fluoro, chloro, bromo, or iodo groups.Preferred are fluoro, chloro or bromo, and more preferred are fluoro andchloro.

“Halogen” means fluorine, chlorine, bromine, or iodine. Preferred arefluorine, chlorine or bromine, and more preferred are fluorine andchlorine.

“Haloalkyl” and “halogenoalkyl” mean an alkyl group as defined abovewherein one or more hydrogen atoms on the alkyl is replaced by a halogroup defined above.

“Ring system substituent” means a substituent attached to an aromatic ornon-aromatic ring system which, for example, replaces an availablehydrogen on the ring system. Ring system substituents may be the same ordifferent.

The term “optionally substituted” means optional substitution with thespecified groups, radicals or moieties.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

An “effective amount” is the dose required to alleviate a particularsymptom of an infection or disease or to protect an animal againstinfections or disease.

As used herein, the term “bovine” refers to animals of the genus Bos,such as cattle. The term “bovid” refers to animals in the familyBovidae, which includes hoofed, hollow-horned ruminants such as cattle,sheep, goats, buffaloes, oxen, etc. As used herein, the term “swine”refers to animals of the family Suidae, which includes pigs, boars,warthogs, etc.

Flunixin meglumine is currently approved globally for use in thetreatment of BRD. It has become a mainstay of veterinary practice forthe treatment of inflammatory conditions. As the veterinarian oftenadministers NSAID therapy with an antibiotic, development of acombination flunixin-antibiotic injectable product would be desirable.Flunixin meglumine is commercially available from, e.g., ISP (Wayne,N.J.), or may be made according to methods known in the art, e.g., themethods described in U.S. Pat. Nos. 3,337,570, 3,478,040 and 3,839,344.

Fluorine-containing analogs of antibiotics chloramphenicol andthiamphenicol have been shown to have antibiotic activity, both againstorganisms sensitive to and resistant to chloramphenicol andthiamphenicol. See Schafer, T. W. et al., “Novel Fluorine-ContainingAnalogs of Chloramphenicol and Thiamphenicol: Antibacterial andBiological Properties,” in CURRENT CHEMOTHERAPY AND INFECTIOUS DISEASEPROCEEDINGS OF THE 11^(TH) ICC AND THE 19^(TH) ICAAC AMERICAN SOCIETY OFM ICROBIOLOGY 1980, 444-446. Examples of such compounds, and methods fortheir manufacture, are described and claimed in U.S. Pat. No. 4,235,892.The medical profession has become increasingly concerned about thetransference of bacterial resistance to humans when antibiotics usefulin treating humans are administered to livestock. Because thechloramphenicol group of antibiotics is infrequently used now to treathumans, its derivatives are particularly appropriate for veterinary use.Of particular interest are the 3-fluoro, 3-deoxy derivatives.

The invention provides novel compositions for the treatment ofinfectious diseases such as bovine respiratory disease in livestock.These compositions are formulations comprising flunixin in combinationwith certain chloramphenicol derivatives. Initial testing of theseformulations demonstrated relatively low serum levels of flunixin incattle following administration of a single dose. In subsequent clinicaltrials, the formulations of the present invention demonstrated anunexpected high degree of efficacy when compared to a negative controlgroup as well as a group receiving florfenicol and flunixin concurrentlybut in separate formulations. The compositions of the present inventioncomprise flunixin meglumine and at least one antibiotic of Formula I:

wherein R is a member selected from the group consisting of methyl orethyl or a halogenated derivative thereof, dihalogenodeuteriomethyl,1-halogeno-1-deuterioethyl, 1,2-dihalogeno-1-deuterioethyl, azidomethyland methylsulfonylmethyl;

each of X and X′ is a member independently selected from the groupconsisting of NO₂, SO₂R₁, SOR₁, SR₁, SONH₂, SO₂NH₂, SONHR₁, SO₂NHR₁,COR₁, OR₁, R₁, CN, halogen, hydrogen, phenyl, and phenyl substituted byhalogen, NO₂, R₁, OR₁, PO₂R₁, CONHR₁, NHR₁, NR₁R₂, CONR₁R₂ or OCOR₁,wherein each of R₁ and R₂ is a member independently selected from thegroup consisting of methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl,isobutyl and phenyl;

and Z is hydrogen or an acyl group of a hydrocarboncarboxylic acid(preferably a hydrocarbondicarboxylic acid) having up to 16 carbon atomsor an acyl group of an amino-hydrocarboncarboxylic acid having up to 12carbon atoms; and the pharmaceutically acceptable salts of said acylgroups.

Included among the halogenated groups contemplated for the moiety R inFormula I are the mono-, di- and tri-fluoro, the mono-, di- andtri-chloro-, the mono- and di-bromo-, and the iodo-methyl groups as wellas the mono- and di-fluoro-, the mono- and di-chloro-, the mono- anddi-bromo-, and the iodo-ethyl groups wherein the halogen substituentsare preferably on the carbon alpha to the carbonyl function. Alsoincluded are mixed dihalogenoalkyl groups in which both halogens arepreferably bonded to the carbon alpha to the carbonyl groups, e.g.,groups such as fluorochloro-, fluorobromo-, and chlorobromo-methyl and-ethyl, as well as trihalogen-methyl groups such as dichlorofluoro- anddifluorochloromethyl.

Also included among the compounds of Formula I are the esterderivatives, e.g. 1-hydrocarboncarboxylates of Formula I wherein Z is anacyl group of a hydrocarboncarboxylic acid having up to 16 carbon atomsthat may be saturated, unsaturated, straight chain or branched chain,aliphatic, cyclic, cyclic-aliphatic, aromatic, aryl-aliphatic, oralkyl-aromatic and may be substituted by hydroxy, alkoxy containing from1 to 5 carbon atoms, carboxyl, NO₂, NHR₁, NR₁R₂, SR₁, SOR₁, or halogen,wherein R₁ and R₂ are as defined above.

Other antibacterially active ester derivatives of Formula I are thosewherein Z is an acyl group of an amino acid containing up to 12 carbonatoms that may be saturated, unsaturated, straight chain, branched chainor cyclic, that may contain aromatic groups and that may be substitutedby hydroxyl groups.

Preferred ester derivatives include those derived from dibasichydrocarboncarboxylates, e.g. the 1-succinate and 1-palmitate esters,which provide water soluble, pharmaceutically acceptable cationic salts,e.g. the sodium or potassium salts as well as salts with amine, e.g.trimethylamine. Also preferred are ester derivatives of amino acids thatprovide water soluble, pharmaceutically acceptable acid addition saltswith mineral or organic acids, e.g. the hydrochloric, or sulfuric acid,or succinic acid addition salts.

As used herein the term “pharmaceutically acceptable salts” thusincludes salts wherein the acidic hydrogen in the dibasichydrocarboncarboxylate esters of this invention is replaced with acation (e.g. sodiumD-(threo)-1-p-nitrophenyl-2-dichloroacetamido-3-fluoro-1-propylhemisuccinate) as well as salts wherein the acidic hydrogen forms anacid addition salt with an amine (e.g.D-(threo)-1-p-nitrophenyl-2-dichloroacetamido-3-fluoro-1-propylhemisuccinate N-trimethylamine salt). Also included are the acidaddition salts formed between mineral or organic acids and the amine inthe amino acid esters of the compounds of Formula I (e.g.D-(threo)-1-p-nitrophenyl-2-dichloroacetamido-3-fluoro-1-propylglycinate hydrochloride).

Among the pharmaceutically acceptable cationic salts of the dibasichydrocarboncarboxylate esters included in Formula I are salts of alkaliand alkaline earth metals (e.g., sodium, potassium, calcium, aluminum)and salts with an amine such as trialkylamines, procaine, dibenzylamine,N-benzyl-beta-phenethylamine, N,N′-dibenzylethylenediamine,N-(lower)alkylpiperidines (e.g. N-ethylpiperidine), and N-methylglucamine.

Preferably R is a halogenated derivative of methyl or ethyl, Z is ahydrogen, X is phenyl, COR₁ or SO₂R₁, R₁ is methyl, and X′ is hydrogen.Most preferably R is CHCl₂ or CHF₂.

A preferred antibiotic compound is florfenicol(D-(threo)-1-p-methylsulfonylphenyl-2-dichloroacetamido-3-fluoro-1-propanol). Another preferredantibiotic compound is D-(threo)-1-p-methylsulfonylphenyl-2-difluoroacetamido-3-fluoro-1-propanol. Processes for themanufacture of these preferred antibiotic compounds, and intermediatesuseful in such processes, are described in U.S. Pat. Nos. 4,311,857;4,582,918; 4,973,750; 4,876,352; 5,227,494; 4,743,700; 5,567,844;5,105,009; 5,382,673; 5,352,832; and 5,663,361.

Formulation efforts on the combination product were directed atmaintaining the desirable pharmacokinetic profile of florfenicolallowing for one-shot administration to cattle with BRD. A formulationcontaining 300 mg/mL of florfenicol and 16.5 mg/mL of flunixin, wasdeveloped and administered at a dose of 40 mg/kg of florfenicol, and adose of 2.2 mg/kg of flunixin meglumine. The data presented demonstratedthat the bioavailability of florfenicol is unaltered (see FIG. 1).Moreover, the flunixin component acted in a clinically equivalentfashion to that of FINADYNE. In a clinical study in which thatformulation was compared to the same formulation with twice as muchflunixin and NUFLOR alone, the benefit of flunixin therapy was clearlyevident. But no incremental benefit of a higher flunixin dose wasapparent (see FIG. 3).

Clinical studies reconfirmed the well-established benefits of flunixinin the treatment of bovine respiratory disease in terms of incrementalimprovement in clinical response, antipyretic response and decreasedlung consolidation. Superiority to antibiotic use alone was evident,particularly in the critical first 24 hours after diagnosis andtreatment (see FIGS. 4, 5 and 6).

In addition to greater convenience and ease of use, it is believed thata single daily subcutaneous administration of a combination product inaccordance with the present invention will promote humane animal care byreducing the number of injections needed to treat animals and providingmore rapid relief of disease symptoms. By reducing the number ofinjections, manpower costs also may be significantly reduced.

In the formulations of the present invention, the concentration offlunixin typically is from about 1 to about 10% by weight, with thepreferred level being at from about 1.5% to about 3.5%, and an even morepreferred level being at least about 1.65% by weight. The concentrationof florfenicol or other antibiotic typically is from about 10% to about50% w/v, with the preferred level between about 20% and about 40% w/v,even more preferred being at least about 30% w/v.

The remaining portion of the formulations of the present invention is apharmaceutically acceptable carrier comprising at least one solvent. Thepharmaceutically acceptable carrier comprises from about 40% to about80% of the formulation.

Florfenicol is generally soluble in aprotic polar solvents such as apyrrolidone solvent, or N,N-dimethylacetamide, N,N-dimethylformamide,DMSO, acetone or glycerol formal. Preferred pyrrolidone solvents areN-methyl-2-pyrrolidone and 2-pyrrolidone. Accordingly, such an aproticpolar solvent (or a combination of such solvents) is preferred for usein formulations of the present invention that contain florfenicol orsimilar antibiotics. Preferably such a solvent is present at about 5% toabout 80% by weight of the formulation. More preferably such a solventis present at about 10% to about 35% of the formulation.

Other pharmaceutically acceptable solvents may be present in theformulations of the present invention. Suitable solvents include water,ethanol, isopropanol, 1,2-propanediol, glycerin, benzyl alcohol,dimethylisosorbide, triacetin, glycol ethers, propylene glycol andpolyethylene glycol (PEG). Particularly preferred solvents include PEGhaving an average molecular weight between about 200 and about 400,triacetin, dimethylisosorbide, ethanol, and water, and combinationsthereof. These solvents may comprise from 0% to about 75% of theformulation. Preferably they comprise from about 15% to about 60%. Morepreferably they comprise from about 40% to about 55% of the formulation.

The addition of one or more of such additional solvents may be desirableto reduce the viscosity of the formulation in order to provide a productwith workable syringeability. Examples of solvents particularly usefulfor adjusting the viscosity of the formulations of the present inventioninclude water, ethanol, isopropanol, propylene glycol,dimethylisosorbide and triacetin, and combinations thereof.

Other inert ingredients can be added to the present composition, asdesired. Such ingredients include preservatives, chelating agents,antioxidants and stabilizers. Exemplary preservatives include methylp-hydroxybenzoate (methylparaben) and propyl p-hydroxybenzoate(propylparaben). Exemplary chelating agents include edetate sodium.Exemplary antioxidants include butylated hydroxyanisole and sodiummonothioglycerol.

In order to prevent degradation of any of the active ingredients in theformulations of the present invention, the addition of at least onestabilizer has been found to be advantageous. Citric acid is a preferredstabilizer.

In order to prepare the composition of the present invention, thevehicle(s) or a portion of the vehicle(s), are added to the compoundingvessel, followed by the remaining excipients and the actives. Themixture is mixed until all solids are dissolved. Additional solvent tobring the composition to final volume may be added if needed. Additives,such as those listed above, may also be included in the vessel and mixedinto the formulation (the order of addition is not critical).

The compositions according to the present invention will generally beadministered to cattle at from about 1 mg to about 100 mg of theantibacterial per kilogram of body weight per day, and from about 0.5 mgto about 5 mg of flunixin meglumine per kilogram of body weight per day.Preferably the compositions of the present invention will beadministered to bovines at from about 20 mg to about 50 mg of theantibacterial per kilogram of body weight. More preferably the dose willbe about 40 mg/kg of the antibacterial. Preferably the compositions ofthe present invention will be administered at from about 1 mg to about 3mg flunixin meglumine per kilogram of body weight.

The compositions according to the present invention will generally beadministered to swine at a dose of from 15 mg to about 100 mg of theantibacterial per kilogram of body weight per day, and from about 0.5 mgto about 5 mg of flunixin meglumine per kilogram of body weight per day.Preferably the compositions of the present invention will beadministered to swine at from about 20 mg to about 50 mg of theantibacterial per kilogram of body weight and about 1 mg to about 2 mgof flunixin meglumine per kg of body weight.

The compositions may be administered once daily or divided into multipledoses. Often only one dose will be sufficient to treat the infection. Insome circumstances one dose followed by a second dose 48 hours laterwill be required to treat the animal. The precise dose will depend onthe stage and severity of the infection, the susceptibility of theinfecting organism to the composition, and the individualcharacteristics of the animal species being treated, as will beappreciated by one of ordinary skill in the art.

The compositions according to the present invention are particularlyuseful for cattle and other bovids, swine, and other large mammals. Inaddition to the treatment of bovine respiratory disease, thecompositions of this invention are also suitable for the treatment ofinfectious diseases associated with inflammation such as swinerespiratory disease, footrot, acute mastitis, pinkeye (infectiouskeratoconjunctivitis), acute pneumonia, metritis and enteritis. Thedosage regimen for treatment of such diseases would be as describedabove.

Mastitis is a complex disease that occurs in lactating females, and isof particular economic importance in dairy cows and goats. Severalpathogenic agents may be involved, including Staphylococcus aureus, E.coli, and Streptococcus. The acute form of mastitis has a sudden onset,the udder is enlarged, hot to the touch and tender usually the affectedanimal will have a fever. If not treated promptly, the udder may bepermanently damaged and milk production decreased or lost.

Currently, acute mastitis is treated with antibiotics,antiinflammatories and oxytocin. The use of the formulations of thepresent invention would be an improvement over presently known methodsof treating mastitis because it would combine one of the most effectiveof the standard treatment combinations into a single, convenientlyadministered formulation.

Pinkeye is an acute infectious disease of cattle, sheep and otheranimals that is characterized by inflammation of the tissues of the eye,characterized by inflammation of the tissues of the eye, accompanied bynasal discharge, lacrimation and copious ocular discharge. Affectedanimals may display extreme discomfort, resulting in a drop in milkproduction; in extreme cases permanent blindness occurs. The disease,which is caused by Moraxella bovis in cattle, is widespread, especiallyamong range and feedlot cattle, and is of great economic importance tothe cattle industry.

Currently, pinkeye is treated by administration of various antibiotics.The use of the formulations of the present invention would be animprovement over presently known methods of treating pinkeye because itwould provide good antibiotic therapy along with an NSAID to reduce theocular inflammation.

Footrot (interdigital phlegmon) is an acute infection of theinterdigital space that occurs throughout the world in both beef anddairy cattle. Fusobacterium necrophorum is the major cause of footrot,although other organisms, including Bacteroides melaninogenicus, can beinvolved. The major symptoms include pain, severe lameness, fever,anorexia, and reduced milk production.

Currently, footrot is treated by antibiotic therapy; recommended therapycan involve treatment for up to five days. The use of the formulationsof the present invention for the treatment of footrot would be animprovement over presently known treatments because it would provide theproven efficacy of florfenicol (with fewer administrations), along withan NSAID to reduce the inflammation caused by footrot and make theanimal feel better.

The compositions of the present invention are also useful for theprevention of these diseases in animals at high risk of developing thosediseases. For example, the presently-claimed compositions can beadministered to cattle at high risk of developing bovine respiratorydisease at the same dosages recommended for treatment of bovinerespiratory disease.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES Example 1 Florfenicol/Flunixin Combination Formulations

Percent (w/v) Percent (w/v) Formulation 1A Formulation 1B Florfenicol300 mg/ml Florfenicol 300 mg/ml Ingredient Flunixin 16.5 mg/ml Flunixin33 mg/ml Florfenicol 30.0 30.0 flunixin meglumine  2.737  5.474N-methyl-2-pyrrolidone 25.0 25.0 propylene glycol 15.0 15.0 polyethyleneglycol 300 QSAD QSAD Density  1.161 g/ml  1.167 g/ml

The N-methyl-2-pyrrolidone, propylene glycol and approximately 90% ofthe PEG 300 required were charged to the compounding vessel and mixedwell. The florfenicol and flunixin meglumine were added with mixinguntil all solids were dissolved. The volume was adjusted with theremaining PEG 300.

Example 2 Pharmacokinetics of Florfenicol in Combination Product

Formulation 1A of Example 1 (SCH 529752) was assessed in a pilotpharmacokinetic study involving 12 cattle in which 6 receivedflorfenicol (NUFLOR) subcutaneously and 6 received Formulation 1Asubcutaneously. A dose of 40 mg/kg of florfenicol and a dose of 2.2mg/kg of flunixin was used. The results are shown in FIG. 1. Thispreliminary study provided good evidence that the pharmacokinetics offlorfenicol, the formulation of the present invention, are very similarto that provided by the already-marketed product, NUFLOR.

Example 3 Pharmacokinetics of Flunixin in Combination Product

In a second study, the pharmacokinetic profile of the second activecomponent in Formulation 1A, flunixin, was examined. The pharmacokineticprofile of flunixin was evaluated following administration to cattle asthe approved single entity flunixin meglumine product, FINADYNE, and asthe combination product Formulation 1A. This study was conducted in fourphases using six cattle (3 males, 3 females). All animals receivedFINADYNE intravenously (IV) in the first phase. This was followed, aftera washout period, by the administration of FINADYNE intramuscularly (IM)to 3 cows and FINADYNE subcutaneously (SQ) to 3 cows. In the next phasethe animals were crossed over and received FINADYNE IM or SQ. In thefinal phase, all six cattle received the flunixin/florfenicolcombination product (Formulation 1A). All animals received flunixin at adose of 2.2 mg/kg, regardless of the formulation or route ofadministration.

Blood samples for determination of flunixin concentration were collectedat 0, 0.17, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12 and 24 hours after dosing.The results are summarized in Table 1 below and in FIG. 2.

TABLE 1 C_(max) T_(max) AUC (0-inf) F Route (ng/mL) (hr) (hr*ug/mL) (%)Flunixin IV — — 13.6 Flunixin IM 3235 0.51 10.5 78 Flunixin SQ 2858 0.5410.9 81 Formulation 1A 1480 1.21 6.47 48 SQ

At the final phase of the study, the 6 cattle were given Formulation 1Aat a dose of 10 mL per 75 kg SQ (equivalent to 40 mg/kg of florfenicoland 2.2 mg/kg of flunixin). The kinetics of flunixin when administeredas Formulation 1A are different from the kinetics of flunixin whenadministered as FINADYNE. As such, the clinical dose response ofFormulation 1A were ascertained in a subsequent study (described below).

Example 4 Clinical Response of Cattle to Flunixin in Combination Product

As the study of Example 3 demonstrated that the bioavailability offlunixin from the combination formulation (Formulation 1A) was lowerthan the approved single-entity product, FINADYNE, a clinical study wasundertaken to compare the clinical response of cattle to flunixin whenadministered at two different concentrations of flunixin withincombination formulations (Formulation 1A and Formulation 1B).

Ninety-six (96) head of cattle demonstrating classic symptoms of bovinerespiratory disease were randomly allocated to treatment with one of thefollowing three treatments at a dose of 20 mL/150 kg of body weight(BW):

TABLE 2 Route of Animals/ Treatment Dosage Administration TreatmentFormulation 40 mg/kg florfenicol & 2.2 SQ 32 1A mg/kg flunixin, SID × 1Formulation 40 mg/kg florfenicol & 4.4 SQ 32 1B* mg/kg flunixin, SID × 1NUFLOR 40 mg/kg florfenicol, SQ 32 SID × 1 *Double the concentration offlunixin in Formulation 1A above

All three treatments were administered subcutaneously once at 40 mg/kgBW of florfenicol (20 mL/150 kg BW). Formulation 1A co-deliveredflunixin at a dose of 2.2 mg/kg BW. Formulation 1B co-delivered flunixinat a dose of 4.4 mg/kg BW. Rectal temperature was assessed at varioustimes throughout the study. Results are shown in FIG. 3. The antipyreticresponse was similar in both treatment groups receiving combinationformulations and superior to NUFLOR at all timepoints. It was concludedthat there was no incremental benefit to increasing the concentration offlunixin with in the combination formulation.

Example 5 Dose Confirmation Study for Flunixin

The optimal dose of flunixin determined in the study of Example 4 wasused in a dose confirmation study. For this study, one-hundredseventy-five (175) beef feeder calves exhibiting clinical signs of acuteBRD, were selected. Enrollment was limited to cattle demonstrating thefollowing clinical signs:

-   -   Clinical illness index score of 2 (moderately ill) or 3        (severely ill)    -   At least two respiratory characteristics scored as abnormal        (polypnea, dyspnea, coughing, mucopurulent nasal discharge)    -   Pyrexia≧104.5° F.        Upon enrollment the cattle were randomly allocated to one of 3        treatment groups:

TABLE 3 Route of Animals/ Treatment Dosage Administration TreatmentFormulation 40 mg/kg florfenicol & 2.2 SQ 26 1A mg/kg flunixin, SID × 1NUFLOR + 40 mg/kg florfenicol & 2.2 SQ (NUFLOR) 30 FINADYNE mg/kgflunixin, SID × 1 and IV (FINADYNE) NUFLOR 40 mg/kg florfenicol, SQ 30SID × 1

The objective of this study was to compare the treatment response to acombination formulation of the present invention relative to an approvedpositive control (FINADYNE and NUFLOR, each given by their approvedroute of administration) and negative (NUFLOR alone) control. Efficacywas assessed by scoring of clinical symptoms, rectal temperature andtotal lung consolidation.

After enrollment and treatment on Day 0, the calves had their rectaltemperatures taken at approximately 2, 6 and 10 hours post-treatment.Calves were then clinically scored once daily on Days 1, 2, 3 and 4;rectal temperatures were taken once daily after the animal's morningobservation.

Treatment success was assessed on Study Day 4. An animal was classifiedas a treatment success if the animal had a rectal temperature of ≦103.5°F., a clinical illness index score ≦1 (slightly ill) and normalrespiratory character (less than two abnormal scores for polypnea,dyspnea, coughing and mucopurulent nasal discharge).

Lastly, on Day 4 the calves were humanely euthanized for necropsy andevaluation of pneumonic lesions/lung consolidation. Percent lungconsolidation was calculated based on visual and palpation scores of 0,1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% assigned to each of theeight lung lobes. The overall lung consolidation is then calculatedusing a standard formula (see Shaw, et al., DISEASES OF SWINE, 8^(th)Ed. 914 (ISU Press, 1999).

Results from this study are summarized in FIGS. 4, 5 and 6.

The addition of flunixin to treatment resulted in a more rapid decreasein rectal temperature (p<0.001 at 2, 6 and 10 hours post-treatment)relative to administration with florfenicol alone. The response was thesame whether the flunixin is administered in a separate syringe andgiven IV or co-administered within a subcutaneous injection ofFormulation 1A (see FIG. 4).

The addition of flunixin to treatment resulted in numerically less lungconsolidation (p=0.08 [Formulation 1A vs. NUFLOR], p=0.1[NUFLOR+FINADYNE vs. NUFLOR]). The response is the same whether theflunixin is administered in a separate syringe and given IV (FINADYNE)or co-administered within a subcutaneous injection of Formulation 1A(see FIG. 5).

As shown in FIG. 6, the addition of flunixin numerically improved therate of success (p=0.1 [Formulation 1A vs. NUFLOR], p=0.3[NUFLOR+FINADYNE vs. NUFLOR]).

Example 6 Florfenicol/Flunixin Combination Formulations

Ingredient Percent (w/v) florfenicol 30.0 30.0 50.0 20.0 flunixinmeglumine 2.74 2.7 4.6 1.8 N-methyl-2-pyrrolidone (NMP) 25.0 — — —citric acid 1.0 — — — propylene glycol 15.0 — — — dimethylacetamide —11.9 30.0 — glycerol formal — QS QS QS polyethylene glycol (PEG) 300 QS— — —

The NMP or dimethylacetamide, citric acid, propylene glycol, andapproximately 90% of the PEG 300 or glycerol formal required for eachformulation were charged to the compounding vessel and mixed well. Theflorfenicol and flunixin meglumine were added with mixing until allsolids were dissolved. The volume was adjusted with the remaining PEG300 or glycerol formal.

Example 7 Florfenicol/Flunixin Combination Formulations

Ingredient Quantity florfenicol 200 mg/ml flunixin meglumine 16.5 mg/mlmethyl p-hydroxybenzoate 1 mg/ml propyl p-hydroxybenzoate 0.1 mg/mlN-methyl-2-pyrrolidone (NMP) 250 mg/ml water 0.15 ml polyethylene glycol200 QS

The NMP and a portion of the polyethylene glycol 200 are charged to thecompounding vessel. Florfenicol, methyl p-hydroxybenzoate and propylp-hydroxybenzoate are added to the vessel and mixed until dissolved.Water is added, followed by the flunixin meglumine, and mixed untilsolids are dissolved. Q.S. to final volume with polyethylene glycol 200if needed.

Although certain presently preferred embodiments of the invention havebeen described herein, it will be apparent to those skilled in the artto which the invention pertains that variations and modifications of thedescribed embodiments may be made without departing from the spirit andscope of the invention. Accordingly, it is intended that the inventionbe limited only to the extent required by the appended claims and theapplicable rules of law.

1. A composition for the treatment of microbial infection in an animal,consisting of about 300 mg/ml of florfenicol, about 16.5 to about 33.0mg/ml of flunixin and/or a pharmaceutically acceptable salt of flunixin,triacetin, 2-pyrrolindone and other inert ingredients wherein the inertingredients are selected from the group consisting of preservatives,chelating agents, antioxidants, and stabilizers.